Carboxylesterase (CE) is one of a sub-class of enzymes known collectively as hydrolases. Hydrolase enzymes catalyze the hydrolysis of various bonds, with carboxylesterase specific for the hydrolysis of both aliphatic and aromatic carboxylic esters, thereby generating an alcohol and a carboxylic acid anion. CEs are ubiquitous serine esterase enzymes that are thought to be involved in the detoxification of xenobiotics, and are found in animal tissues (primarily the liver, serum, lung, kidney, intestine, and blood brain barrier), plants, molds, and yeast. The tissue distribution of these enzymes correlates with their involvement in xenobiotic detoxification.
As yet, no endogenous substrates for CEs have been identified, although they are responsible for the metabolism of many drugs, including CPT-11, cocaine, heroin, meperidine, and capecitabine. These carboxylesterase enzymes are processed in the endoplasmic reticulum of mammalian cells, and hence these proteins can be secreted into the extracellular milieu. Recently, the x-ray crystal structure of a rabbit-liver and a human-liver carboxylesterase have been determined. These studies indicate that the proteins demonstrate similar structures to other esterases including acetylcholinesterases, lipases, etc.
Previously known inhibitors of esterases have included organophosphorous compounds such as di-isopropyl fluorophosphate (DFP), carbamates, piperidine derivatives, and acridine derivatives. These compounds are regarded as highly toxic poisons. The inhibition of chicken liver carboxylesterase by benzil has been reported in a study of the enzymatic mechanism involved. However, the only selective inhibitors of CEs that have been reported are the cyclic organophosphate derivatives, Bomin-1, 2, and 3 (Latoxan, France).
7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (Irinotecan, CPT-11) is a widely used anti-cancer drug that has demonstrated remarkable promise in the treatment of solid tumors. CPT-11 has demonstrated remarkable antitumor activity in both preclinical models and patients with refractory disease and, as such, has recently been approved for the treatment of colon cancer in adults. When administered to patients, CPT-11 is activated by human carboxylesterase to yield its active metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38), which is a potent topoisomerase I poison. Topoisomerases are the enzymes responsible for unwinding and winding chromosomal DNA. In order to allow transcription and translation, DNA must be unwound. SN-38 prevents DNA unwinding, and thus inhibits critical cellular processes in tumor cells, resulting in cell death.
The toxicities associated with this agent include a cholinergic syndrome due to direct inhibition of acetylcholinesterase, and delayed diarrhea due to gastrointestinal toxicity. The gastrointestinal toxicity is thought to occur via two independent mechanisms:
1. SN-38 is conjugated in the liver to yield SN-38 glucuronide (SN-38G). Following deposition into the small intestine via the bile, SN-38G can be cleaved by bacterial glucuronidases to yield the toxic metabolite SN-38, resulting in local irritation and toxicity to the gut.
2. CPT-11 is also eliminated via the bile, and following entry into the gut, CEs present within the intestinal epithelia can convert the drug to SN-38. Hence very high, local concentrations of SN-38 will be produced, resulting in cytotoxicity and hence diarrhea.
The delayed diarrhea associated with CPT-11 administration can be life-threatening and is the dose limiting toxicity for this agent. Potential solutions for ameliorating this toxicity include: (i) the aggressive use of antidiarrheals, such as loperamide and diphenoxylate/atropine, and (ii) the alkalinization of the gut using bicarbonate.
The level of activation of CPT-11 by human plasma in vitro is very low. In contrast, plasma derived from rats and mice is very proficient at CPT-11 activation, with greater than 50% of the drug converted to SN-38 within 1 hour of incubation. Either the levels of the enzymes responsible for CPT-11 metabolism in humans are low, or these human proteins have significantly diverged in structure from their rodent counterparts. Hence, animal models designed to predict tumor responses in humans may overestimate the efficacy of the drug due to the increased plasma activation of CPT-11.
Recently, a rabbit liver carboxylesterase that could efficiently convert CPT-11 to SN-38 was isolated. A human homolog of this carboxylesterase (hCE1) is known. However, expression of hCE1 in human tumor cells does not alter their sensitivity to CPT-11. More recently, it has been demonstrated that both the human and mouse small intestine expresses high levels of carboxylesterases that can convert CPT-11 to SN-38. A cDNA encoding a human small intestinal carboxylesterase (hiCE) has subsequently been identified that is highly efficient at activating CPT-11. Expression of this protein in mammalian cells sensitizes them to the drug.
Therefore, it appears that the activation of CPT-11 in the human intestine by hiCE results in local toxicity, and hence produces the unwanted toxic side effects such as diarrhea. Therefore, before CPT-11 and similar anti-cancer drugs can be utilized to their full potential, there is a need to develop methods for alleviating the toxicity problems associated with administration of these drugs.
There is therefore a need to develop new compounds that are not only useful as general esterase inhibitors, but further, to develop new compounds that are specific for the inhibition of selected carboxylesterases, such as the human small intestine carboxylesterases (hiCEs) that activate drugs such as CPT-11.
The following patents and publications provide relevant background to the present invention. All references cited below are incorporated herein by reference in their entirety and to the same extent as if each reference was individually incorporated by reference. U.S. Pat. Nos. 5,762,314, 6,407,117; Published International Application No. WO 99/42593; Khanna et al., Cancer Research, 2000, 60: pp. 4725-4728; Wadkins et al., Molecular Pharmacology, 2001, 60(2): pp. 355-362; Wierdl et al., Cancer Research, 2001, 61: pp. 5078-5082; Tanizawa et al., J. Natl. Cancer Inst., 1994, 86: pp. 836-42; Morton et al., Mol. Biotechnol., 2000, 16, pp. 193-202; Soares, E. R., Biochem. Genet., 1979, 17, pp. 577-583; and Berndt et al., Biochimica et Biophysica Acta., 1996, 1298, pp. 159-166.